As complete genome sequences of intracellular bacterial symbionts and pathogens accumulate, an emerging trend is that transposable insertion sequence (IS) elements often proliferate after bacteria transition from a free-living to an intracellular lifestyle. IS elements are generally detrimental. However, they add inherent fluidity to their hosts'genomes, particularly when they are abundant. Because intracellular bacteria are essentially asexual, profuse IS elements may be an important source of genetic variation for these populations. This underscores the importance of elucidating the evolutionary dynamics of IS elements within intracellular symbionts and pathogens. The objective of this project is to test the following two hypotheses for why IS elements often expand in intracellular symbionts: (1) intracellular bacteria have relatively small population sizes, so natural selection is less effective at purging deleterious genetic changes because stochastic genetic drift plays an increasingly important role in these populations, and (2) intracellular symbionts are often bathed in a nutrient-rich environment and do not have to synthesize many of their own required metabolites, so many biosynthetic genes may be essentially selectively neutral territory for IS element insertion. These hypotheses will be tested using an experimental evolution approach with E. coli. In short, replicate E. coli populations (which have 44 resident ISs) will be subjected to four experimental treatments for 5000 generations: (1) large population size/rich medium, (2) small population size/rich medium, (3) large population size/minimal medium, and (4) small population size/minimal medium. If IS elements spread significantly more in small populations than large populations, then this will support the hypothesis that ISs expand when natural selection is relaxed due to pronounced genetic drift in small populations. Alternatively, if IS elements spread significantly more in nutritionally rich medium populations than minimal medium populations, then this will support the hypothesis that relaxed selection on superfluous biosynthetic genes allows ISs to proliferate. These two hypotheses are not mutually exclusive and both could be important in shaping IS expansion in intracellular symbionts. If so, then IS expansion will negatively correlate to population size for each media type, with the minimal media values lower than the rich media values. Project Narrative: IS elements add inherent fluidity to their hosts'genomes (e.g., Parkhill et al. 2003;Song et al. 2004;Yang et al. 2005). Therefore, their frequent proliferation in intracellular pathogens may be fundamentally important in generating genetic variability in bacteria that rarely encounter and exchange DNA with other bacteria. This underscores the importance of elucidating the evolutionary dynamics of IS elements within intracellular pathogens.